The present invention relates to devices comprising electrosensors containing capture reagents, their preparation thereof, and their use for detecting, preferably, quantitative measurement, of analyte in a liquid sample. In particular, the invention relates to an enzyme electrosensor, e.g., electroimmunosensor, device for electrochemical detection and preferably, real-time measurement, which is suitable for use at point-of-care settings by unskilled personnel.
There is an increasing public awareness of the need for diagnostics to determine levels of various components in human fluids, such as blood or serum. Of particular interest are tests designed for non-expert use that produce rapid and quantitative results.
Immunoassays have been widely used for the detection of antigens and antibodies. The most commonly used immunoassays are enzyme immunoassays (EIAs). The importance of EIAs, particularly in clinical analyses, medical diagnostics, pharmaceutical analyses, environmental control, food quality control, and bioprocess analyses, lies in their high sensitivity and specificity, which allow the detection of a wide spectrum of analytes in various sample matrices.
EIAs are commonly referred to as either heterogeneous (necessitating free antigen separation from those that have been bound to antibody) or homogeneous (requiring no separation or washing steps during the assay). Also, EIAs can be either competitive or non-competitive, depending on the availability of antibody binding sites. Conventional EIAs are convenient for analysis of great numbers of samples on a routine basis and are widely used in a broad spectrum of applications. However, these methods require multiple washing and incubation steps to implement, and can be utilized in high volume only by complex and expensive analytical equipment. The need for multiple washing and incubation steps has also limited the development of portable point-of-care analytical devices that can be used to perform assays in decentralized locations.
In recent years, efforts have been made to overcome the limitations of heterogeneous EIAs and to search for homogeneous, rapid, and separation-free immunoassays that can be readily conducted at the point of care. Fast and simple EIA tests capable of detecting a single analyte with a color change that can be visually interpreted have been developed. Based on the techniques of immobilizing antigen or antibody on a solid-phase support, assay formats such as dipsticks, test tubes, and wicking membrane test cartridges have been used to provide fast results for analytical conditions where a simple qualitative (yes/no) answer is clinically relevant. These membrane-based assays have gained increasing popularity in many areas of clinical chemistry. They not only form the basis of the majority of home use tests, but also are rapidly gaining use in the physician""s office and hospital lab. These tests are widely accepted and increasingly used for detection of pregnancy, strep throat, and bacteria, as well as for prediction of ovulation. Examples of such assays are described in U.S. Pat. Nos. 5,622,871, 4,703,017, 5,468,647, 5,622,871, and 5,798,273. However, most of these rapid tests are incapable of performing sensitive and quantitative detection. As a result, medical diagnoses that require quantitative measurement of the target analyte remain within the domain of the complex immunoassay analyzers in the centralized laboratory.
A major trend in the development of rapid immunoassays is the move toward quantitative testing. The use of membrane-based immunoassays has been proposed for quantitative measurement of analytes. As a specific example, U.S. Pat. No. 5,753,517 describes a quantitative immuno-chromatographic assay utilizing antibody-coated particles, independent control particles, and capillary flow through a membrane. However, there are difficulties in developing such quantitative immunoassays based on membrane format for point-of-care diagnostic tests. Perhaps the most significant problems with the use of membrane-based immunoassays arise from requirements for the membrane that are contradictory. For example, immobilization of protein in the detection area requires that the membrane have a strong binding affinity for the protein, but transport of analyte and particles containing detection components demands that the membrane not bind to protein. Furthermore, factors commonly used for increasing the performance of the membrane assay are often mutually exclusive. For example, blocking reagents that reduce nonspecific interactions usually also reduce the amount of specific signal. In light of these competing requirements commonly seen in efforts to develop membrane-based immunoassays, it becomes clear that conventional membrane systems have limited advantages for use in quantitative immunoassays.
Accordingly, there is a need to develop improved assays, e.g., immunoassays, that can provide rapid, quantitative, and reliable results. The high sensitivity of electrochemical detection coupled with the inherent specificity of antibody-antigen reactions has resulted in a remarkable technique known as electrochemical immunoassay. The advantages of such assays include, among others, the ability to measure untreated samples in the presence of possible interfering substances, as well as the simplicity, and sensitivity associated with electrochemical detection.
Immunoassays employing amperometric electrochemical detection have been applied to the determination of analytes in fluid samples. An immunoassay device using amperometric detection to perform diagnostic tests for analytes in body fluids is described, as a specific example, in U.S. Pat. No. 5,830,680. The device includes an electrochemical detection system for a separation-free sandwich-type immunoassay, in which a protein analyte such as human chorionic gonadotropin (hCG) is sandwiched between a capture antibody immobilized on a microporous membrane gold electrode and an alkaline phosphatase-labeled antibody. Although such a device offers a separation-free feature, the time required for manipulating and incubating the sample limit the use of such assays for rapid diagnostic testing.
A method employing liposomes for signal production and electrochemical detection in immunoassays is described in U.S. Pat. No. 5,756,362. In these assays, liposomes that encapsulate an electroactive marker are conjugated with an analyte. A test device first allows incubation of a sample containing an analyte with a binding material specific for the analyte and the analyte-liposome conjugate. Following incubation, the mixture solution is allowed to traverse through an absorbent material strip to reach an electrochemical measurement portion where the liposome is lysed by a lysing reagent to release the electroactive marker. The amount of marker released is then detected electrochemically and correlated with the amount of analyte in the sample.
In the methods described in U.S. Pat. No. 5,391,272, bioactive components are coated onto colloidal gold and subsequently coated onto a sensor. Detection of analyte is achieved by measuring current generated by an electroactive species bound to the sensor as part of an analyte/enzyme catalytic response. Although the method is suitable for detecting several types of analytes (e.g. hormones or herbicides), it involves separation and incubation steps in order to achieve desirable sensitivity.
Other immunoassays using electrochemical detection have to rely on methods conventional in heterogeneous immunoassays, such as lengthy incubation time and multiple washing steps to separate free antigen and detection reagent from bound ones. Although several groups have reported methods for performing non-separation amperometric immunoassays, to date there have been no reports describing an amperometric immunoassay that is simple, rapid, and does not require a separation step.
Accordingly, there is still a need in the art for assay devices and methods that provide simple, quantitative and real time diagnostic measurements. The present invention addresses this other related needs in the art.
The present invention overcomes many of the problems in the art by providing a simple, rapid, and reliable means to measure, and preferably, quantitatively measure, analyte in a liquid sample using a combination of electrochemical detection and binding between the analyte and its capture reagent.
In one aspect, the present invention is directed to a device for detecting an analyte in a liquid sample, which device comprises: a) a solid support; b) an electrosensor immobilized on said solid support, said electrosensor comprises a working electrode and another electrode used as auxiliary and/or reference electrode; c) a capture reagent immobilized on said working electrode, said capture reagent is capable of binding to an analyte; and d) conductive leads for connecting said electrodes to a readout device for electrochemical measurement.
The invention electrosensor, e.g., electroimmunosensor, utilizes a sensor assembly (i.e., sensor strip). The sensor strip can comprise a base sensor, e.g., a sensor fabricated by screen-printing of conductive materials onto a suitable support. A capture reagent that is capable of binding to the analyte is immobilized on the working electrode surface. Preferably, the capture reagent is capable of specific binding to the analyte, which can be either an antibody specific to an epitope of the analyte of interest, or the analyte of interest itself.
In addition to the sensor assembly, the device can optionally has a sample application area and/or a detection area. The detection area covers the region of the electrode surface upon which the capture reagent, e.g., antibody, is immobilized. The application area can include an application pad having a detection reagent pre-immobilized thereon. The detection reagent may be a detection reagent, e.g., an antibody, labeled with an enzyme that is able to produce an electrochemical detectable signal when reacting with substrates.
The sensor assembly can also include a wicking member, e.g., in the form of a strip, that connects the application area and the detection area. The wicking member functions as a carrier or wicking reagent to deliver the fluid sample containing the analyte and the detection reagent through capillary action to the detection area where they become immobilized on the electrode surface through, e.g., antibody-antigen reaction. Examples of materials useful as the wicking member include nylon, cellulose, paper, and the like. A preferred wicking member is a nylon mesh that has open mesh structure. A mesh structure is particularly useful because it has a two-dimensional structure suitable for lateral delivery of the liquid sample from the application area to the detection area to be in direct contact with the capturing reagent.
The sensor assembly may additionally include a conjugate releasing pad for absorption and controlled release of a conjugate and/or a separation filter for separating plasma from whole blood, resulting in the plasma wicking laterally.
An absorbent material used as waste reservoir can be positioned at the end of the sensor assembly, and overlaps with the wicking member to facilitate the migration of the sample through the device surface. The absorbent pad will have sufficient porosity and volume to retain a liquid sample on which the assay is to be performed.
In another aspect, the present invention is directed to a method for assaying an analyte in a liquid sample, which method comprises: a) contacting a liquid sample containing or suspected of containing an analyte with the above-described device under suitable conditions whereby the analyte, if there is any, binds to the capture reagent immobilized on the working electrode and the binding between the analyte and the capture reagent causes a change in the current signal that is capable of being detected by the electrosensor of the device; and b) detecting the change in the current signal generated in step a), whereby the presence or amount of the analyte in the sample is assessed.
In a preferred embodiment, the present method is used to perform an enzyme immunoassay using the invention electroimmunosensor. According to such a preferred method, a sample containing the analyte of interest is applied to the sample application area. The sample is allowed to flow through the membrane assembled in the sensor strip to react with the antibody immobilized on the sensor surface and with the antibody enzyme conjugate. Under appropriate conditions, the analyte is sandwiched between the antibody immobilized on the sensor surface and the antibody conjugate. The amount of analyte in the fluid sample is proportional to the amount of analyte immobilized on the sensor by this process through antibody-antigen interaction and can be detected through the antibody-enzyme conjugate that is bound to the sensor surface through the analyte. The amount of analyte is then determined from a standard curve for the analyte of interest.
Detection can be achieved according to the invention method by coupling the capture reagent-analyte binding reaction, e.g., immunological reaction, if the capture reagent used is antibody, to an electrode response using the enzyme conjugated to the analyte specific reagent as indicator. The current generated from a sensor assembly under controlled conditions is proportional to the analyte concentration present in the fluid sample and can be measured using a electrocurrent detection device, e.g., an amperometric monitor. Detection of analytes in buffer and serum samples can be accomplished using the invention electrosensors, e.g., electroimmunosensors, in either competitive or sandwich assay format.
Invention methods yield a rapid result by applying a test sample to a disposable sensor strip and initiating electrochemical detection when the invention electrosensor is connected to an electrochemical instrument, such as a hand-held detector. Good sensitivity and quantitative results can be achieved within minutes by unskilled personnel at point-of-care settings. Furthermore, the methods can generally be used for quantitative measurement of virtually any analytes, especially and immunologically active species, in a liquid sample, thus providing broad application in medical diagnostics and prognostics, and in agricultural and environmental assessments. In addition, the electrosensor can be provided in a kit suitable for use at home, in a physician""s office, or in other point-of-care settings.
In still another aspect, the present invention is directed to a method for preparing an electrochemical sensor for the detection of an analyte in a liquid sample, which method comprises immobilizing a capture reagent capable of binding to an analyte on the surface of a hydrophobic, non-metal electrode by contacting said electrode surface with a solution containing said capture reagent and an organic immobilizing agent that wets said electrode surface and facilitates immobilization of said capture regent on said electrode surface.
In yet another aspect, the present invention is directed to a kit for detecting an analyte in a liquid sample, which kit comprises: a) a device comprising a solid support, an electrosensor immobilized on said solid support, said electrosensor comprises a working electrode and another electrode used as auxiliary and/or reference electrode, a capture reagent immobilized on said working electrode, said capture reagent is capable of binding to an analyte, and conductive leads for connecting said electrodes to a readout device for electrochemical measurement; and b) an effective amount of a suitable electron transfer mediator and substrate, and any other buffer solutions, conjugate solutions or, standards necessary for performing the detection assay.
In yet another aspect, the present invention is directed to a device for detecting an analyte in a liquid sample, which device comprises a sample application area that is in fluid communication with an electrosensor via a wicking member, wherein the wicking member has an open mesh structure.